Mass spectrometry (MS) is a powerful, widely used technique for the qualitative and quantitative analysis of chemical substances ranging from small molecules to macromolecules. In general, it is a very sensitive and specific method, and it comes in many forms, allowing gases, liquid and solid samples to be analyzed. Many of the samples are biomedical or environmental.
Among the major limitations that have held back the general usefulness of mass spectrometry, however, three are relevant here. The first limitation is that the MS signal tends to be analyte dependent, which means that some analytes are much more sensitive than others when detected by this technique. The second problem is background chemical noise, which comes primarily from the sample and often prevents high sensitivity (the ratio of signal to noise). Such noise typically is worse when the amount of analyte is lower or when the analyte is derived from a complex biological or environmental sample as opposed to having its origin as a standard. The third problem, which is related to the first, is that the optimum conditions in the mass spectrometer for high sensitivity tend to depend on the analyte.
Some analytes inherently are more sensitive than others to detection by tandem mass spectrometry because they readily form a gas phase parent ion in the mass spectrometer. For other analytes, the parent ions can undergo a favorable dissociation when activated energetically, as in a collision-induced dissociation step, to form a favorable daughter ion which may be detected relatively free of noise. A relatively intense daughter ion can arise when the compound naturally contains a bond that preferentially dissociates upon energetic activation. An example of this second property is the sensitive detection of a drug containing a labile piperidinyl-allyl bond by MS/MS (Dear et al., 1999). However, many analytes lack either of such favorable properties for sensitive detection by MS/MS.
A common strategy in mass spectrometry to increase signal strength for a poorly-responding analyte is to covalently tag the analyte with a signal-enhancing molecular group. For example, a cationic tag can be employed to enhance the signal for an analyte in an electrospray mass spectrometer or a laser desorportion mass spectrometer including the technique of matrix assisted laser desorption ionization (Zhao et al., 1997). As a second example, (2-hydroxyethyl)trimethylammonium chloride (choline chloride), a saturated compound (lacking double or triple bonds), was used to derivatize fatty acids after the latter were converted to acid chlorides, and the products were detected in a tandem mass spectrometer (Johnson, 2000). Because the choline chloride tag lacked a reactivity group, it was necessary to create an acid chloride reactivity group on the analyte. A comparison of the trimethylammonium and dimethylamine derivatives showed that the former were more prone to detection by neutral loss (of trimethylamine). The application of the method to hydroxy fatty acids was especially complicated by the need to protect the hydroxy group on these acids by acetylation. Additional fragment ions also formed by neutral loss from the hydroxy fatty acids, further compromising the sensitivity. Moderate sensitivity at best was achieved.
Chemical tags have been disclosed with reactivity groups that were reacted with peptides for the purpose of enhancing the formation of sequence-specific daughter ions (Zaia et al., 1995). A complication reported by these authors was that some of the tagged peptides lost a neutral fragment from the tag. These neutral-loss cleavages were considered to be a nuisance since they competed with cleavage along the peptide chain. The latter cleavages were desired since they are the ones that provided sequence information. In most cases, the abundance of the analyte-characteristic daughter ion from neutral loss was less than 1% of the intensity of the parent ion, but in one case it was reported to be 20.9%. Since the relative abundance of the peptide ions from neutral loss was relatively low, even in the latter case, Zaia and Biemann stated, in regard to the ions from neutral loss, “These ions are not sufficiently abundant to detract from the quality of the spectrum.” Of the tags studied, one that minimized neutral loss was recommended. In a review on the subject (Roth et al., 1998), it was similarly stated, “The added chemical derivative group should not fragment during analysis by tandem mass spectrometry, because that fragmentation might complicate the mass spectrum and reduce the intensity of the other fragment ion peaks.” Thus, the best tags for the purpose of enhancing peptide sequencing by tandem MS have been considered to be the ones with minimal fragmentation characteristics. The general use of phosphonium tags to enhance sensitivity for mass spectrometry was also covered in this review.
Thompson et al. (2003) have introduced the concept of a molecular tag termed a “tandem mass tag” (TMT) for quantifying relative amounts of protein-derived peptides in two samples by means of tandem mass spectrometry. The relative quantitation is based on measuring the terminal charged group (sensitization group) of the tag that is released from the tagged peptide upon collision in the collision-induced-dissociation part of the mass spectrometer. It is, therefore, important for the method of Thompson et al. that the released part of the tag is lost as an ion rather than as a neutral fragment, so that it can be detected efficiently in the mass spectrometer. Pairs of tags differing in terms of isotope distribution are used, where each member of each pair of tags has the same overall content of isotopic atoms, but a different location of these atoms. One member of each pair of tags is to be used separately in a non-combined way for the tagging step for each peptide sample to be compared one against the other. After this tagging step, the two samples are combined. The detected ions for relative quantification, which are tag-derived, would be the same for every pair of peptides in the two samples being compared. Thus, these relative quantification ions provide no qualitative information about the peptides, such as their masses. There is also the option of detecting the residual peptide part based on the charge provided to it by protonation.
Finally, aryl sulfates can readily lose a SO3 neutral fragment in the collision cell of a tandem mass spectrometer. For example, Zhang et al., (1999), observed that the base peak in the tandem mass spectrum of several estrogen sulfates, in which the sulfate is attached to the phenyl ring, came from loss of SO3 from the parent anion. The sulfo group was similarly observed to be labile in sulfotyrosine peptides analyzed in a tandem mass spectrometer (Rappsilber et al., 2001). However, no tags were employed having an aryl sulfate moiety.
Thus, there is still need for improved analyte tags for mass spectrometry that would consistently provide increased sensitivity. In addition, a category of tags that were not dependent for their performance on characteristics of the analyte would be particularly desirable.